Bifidobacterium breve ferm BP 6223 for producing fermented soymilk

ABSTRACT

A  Bifidobacterium breve  strain provided is showing a viable count of 1×10 9  cells/ml or more immediately after culturing in a soymilk medium and sustaining a viable count of 1×10 8  cells/ml or more after storage at 10° C. for 14 days. The strain is preferably  Bifidobacterium breve  FERM BP-6223, and is used for producing fermented soymilk.

TECHNICAL FIELD

The present invention relates to Bifidobacterium breve which excellentlysurvives storage, and to fermented soymilk of pleasant flavor containingthe same.

BACKGROUND ART

Since long ago, fermentation of soymilk by use of microorganisms hasbeen carried out in order to attain sensory improvement of soymilk,which has a characteristic bean odor and grassy taste. In the course ofsuch fermentation treatment, a variety of microorganisms such as lacticacid bacteria, yeasts, Bacillus natto, acetic acid bacteria, andAspergillus have been employed. It is reported that in most cases suchtreatment yields fermented products having pleasant flavor.

Soymilk has been known to promote proliferation of microorganisms whichbelong to genus Bifidobacterium (hereinafter referred to asbifidobacteria), possibly due to soybean oligosaccharide contained insoymilk (Yoichi KOBAYASHI et al., “Intestinal Flora and Food Factors,”p69, 1984, published by Gakkai Shuppan Center).

Meanwhile, a number of studies have been conducted on usefulness ofbifidobacteria. For example, administration of a live-bacteriumpreparation (Bifidobacterium breve) has proven to exhibit a curativeeffect on refractory pediatric diarrhea, and a possible mechanismthereof may be attributed to improvement of intestinal flora.Furthermore, it has been found that administration of the preparation toan adult results in reduction of putrefied products in feces andputrefied-product-producing bacteria. In addition, an immunoactivatingeffect of bifidobacteria has also been elucidated. Thus, bifidobacteriaare considered to contribute to human health through improvement of theintestinal environment (edited by Tomotari MITSUOKA, “Study ofBifidobacteria,” 1994).

Thus, a variety of products employing bifidobacteria have beendeveloped, and simultaneously efforts have been made to develop afermented soymilk product which contains numerous bifidobacteria andwhich also maintains a viable count (high survival rate) throughout theperiod required for product distribution.

However, since bifidobacteria generate lactic acid and acetic acid at amole ratio of 1:1.5 through metabolism of sugar, bifidobacteria tend toperish by self-produced acid. Therefore, growth thereof to a certainviable count or higher and maintenance of the viable count aredifficult.

In order to solve the aforementioned problem, employment of abifidobacterium, of high survival rate has been proposed (JapanesePatent No. 2563197).

However, even though the employed bifidobacterium shows an improvedsurvival rate as compared with that of a conventional bifidobacterium,the employed bacterium cannot be proliferated to a level of 1×10⁹cells/ml or higher, and maintenance of a viable count of at least 1×10⁸cells/ml after 14 days' storage is difficult.

Thus, an object of the present invention is to provide a bifidobacteriumwhich can sustain a certain level of viable count even after storage fora certain period of time. Another object of the invention is to providefermented soymilk containing the bifidobacterium.

DISCLOSURE OF THE INVENTION

In view of the foregoing, the present inventors have conducted earneststudies, and have discovered a bifidobacterium satisfying theaforementioned requirement for viable count and have also found thatfermented soymilk of pleasant flavor and high viable count can beproduced by use of the bifidobacterium. The present invention has beenaccomplished on the basis of these findings.

Accordingly, the present invention provides Bifidobacterium breveexhibiting a viable count of 1×10⁹ cells/ml or more immediately afterculturing in a soymilk medium and sustaining a viable count of 1×10⁸cells/ml or more after storage at 10° C. for 14 days. The invention alsoprovides fermented soymilk containing the same.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing survival of bacteria contained in soymilk.

FIG. 2 is a graph showing survival of the YIT 4065 strain in fermentedsoymilk.

BEST MODES FOR CARRYING OUT THE INVENTION

The Bifidobacterium breve of the present invention exhibits a remarkablyhigh survival rate in a soymilk medium and, after being grown to a levelof 1×10⁹ cells/ml, sustains a viable count of 1×10⁸ cells/ml or moreeven after storage at 10° C. for 14 days.

Example microorganisms which belong to Bifidobacterium breve include amicroorganism named YIT 4065 and deposited as FERM BP-6223 (originaldepository date: Feb. 29, 1996) to the National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology(address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305-0046 JAPAN)in accordance with the Budapest Treaty. The bacterium is produced in thefollowing manner.

Cultured cells (1.5×10⁹ cells) of Bifidobacterium breve YIT 4006deposited as FERM BP-752 (original depository date: Jan. 28, 1977) tothe Fermentation Research Institute, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry (address: 1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305-0046 JAPAN) in accordancewith the Budapest Treaty) are suspended in 50 mM phosphate buffer (pH7.0), and an equiamount of a 5 μg/ml solution of NTG(N-methyl-N′-nitro-N-nitrosoguanidine) is mixed with the suspension. Theresultant mixture is maintained at 37° C. for 30 minutes. After removalof NTG by washing, the resultant mutation-treated cells are inoculatedto a 14% whole milk powder medium (yeast extract content: 0.03%) andcultured at 37° C. until the pH reaches 4.5-4.7. Fine liquor is added tothe cultured product in an amount of 8%, and the mixture is poured intotest tubes. Each test tube is sealed with a butyl rubber stopper andstored at 10° C. The viable count is measured over time by use of amodified Rogosa agar medium. When the viable count reaches about 1%, thestored product is inoculated again to a 14% whole milk powder medium(yeast extract content: 0.03%) and cultured at 37° C. until the pHreaches 4.5-4.7. After addition of sugar to the cultured product, themixture is stored again. This procedure ofstoring-counting-inoculation-culturing is repeated four times, and fromthe thus-obtained fermented product, a strain showing improved survivalis separated as Bifidobacterium breve YIT 4065.

In addition to the aforementioned survival property, Bifidobacteriumbreve YIT 4065exhibits the following mycological properties.

The bacterium is proven to be pleomorphic; i.e., rod-like or branching,through observation under an optical microscope, and is gram-positive.When the bacterium is anaerobic-cultured on a modified Rogosa agarmedium, a white and glossy colony is formed, whereas when it issimilarly cultured on a milk medium, lactic acid and acetic acid areformed to curdle milk. Catalase activity is negative.

The bacterium exhibits positive fermentation activity to ribose,fructose, galactose, sucrose, maltose, cellobiose, lactose, melibiose,raffinose, melezitose, and salicin and negative activity to arabinose,xylose, trehalose, mannitol, sorbitol, and inulin.

The fermented soymilk of the present invention can be produced throughfermentation of soymilk by use of the aforementioned Bifidobacteriumbreve.

The soymilk serving as a raw material in the present invention ispreferably produced from untreated soybeans containing oils and fats,defatted soybeans, soybean flakes, etc. Defatted soybeans may serve as araw material.

Soymilk can be produced by dipping raw material in water; crushing withaddition of hot water optionally containing 0.5-1.0 wt.% (hereinaftersimply referred to“%”) of sodium carbonate; removing okara (bean-curdrefuse); and sterilizing with heat. However, production of soymilk isnot limited to this method, and the soymilk used in the presentinvention may be produced in any manner.

To the soymilk, there may be added nutrients required for growingbacteria, such as sugar incorporated into food, e.g., sucrose, glucose,fructose or invertose; meat extract; peptone; yeast extract; and peptidein order to carry out post-treatment with a bacterium. In addition, inorder to realize the optimal pH for a target bacterium, acidsincorporated into food such as citric acid, malic acid, ascorbic acid,lactic acid, and acetic acid may be added to the soymilk.

No particular limitation is imposed on the method of fermentation, andfermentation may be carried out under typical conditions. For example,when Bifidobacterium breve YIT 4065 is inoculated to the aforementionedsoymilk, culture may be carried out at 37° C. for approximately 20-22hours until the pH reaches approximately 4.5-4.7. Fermentation may becarried out under conditions appropriately selected in accordance withculture conditions, such as standing culture, spinner culture, shakingculture, and aerobic culture.

Although the fermented soymilk of the present invention may be producedby adding Bifidobacterium breve to soymilk, other bifidobacteria orother microorganisms may further be added. Examples of suchmicroorganisms include bacteria such as Lactobacillus, Lactococcus,Streptococcus, Leuconostoc, Bacillus, Acetobacter, and Gluconobacter;yeasts such as Saccharomyces, Candida, Rhodotorula, Pichia,Schizosaccharomyces, Torula, and Zygosaccharomyces; and filamentousfungi such as Aspergillus, Eurotium, Monascus, Mycol, Neurospora,Penicillium, and Rhizopus.

The thus-obtained fermented soymilk of the present invention may beserve as food as is or may be formed into an oral pharmaceutical, andmay further contain any customarily-employed additive. Examples of suchadditives include saccharides, proteins, fats and oils, vitamins, plantextract, animal extract; bacterium extract; perfumes, and colorants.Furthermore, the fermented soymilk of the present invention may beprocessed into any form, such as liquid, paste, sponge, or solid. Inaddition to a soymilk beverage, the fermented soymilk may be processedinto a variety of food products such as soymilk yogurt, soymilk pudding,and tofu (soybean curd).

EXAMPLES

The present invention will next be described in more detail by way ofexamples.

Example 1

Soymilk (product of Shikoku Kakoki, solid content 12.0%, crude fat2.48%, crude protein 4.71%) was sterilized with steam at 100° C. for 90minutes.

Subculture was carried out several times by use of soymilk producedunder anaerobic conditions, to thereby produce two Bifidobacterium breveinocula and two Bifidobacterium longum inocula shown in FIG. 1. Each ofthese inocula was inoculated to the aforementioned sterilized soymilk inan amount of 0.5-2%, and cultured until the pH reached 4.6.

After addition of glucose to the fermented product in an amount of 5%,the mixture was stored at 10° C. under anaerobic conditions. Viablecells were counted, and the results are shown in FIG. 1.

Both Bifidobacterium breve samples showed a viable count of 1×10⁹cells/ml immediately after preparation of a fermented product. However,Bifidobacterium breve ATCC 15700 failed to sustain a viable count of1×10⁸ cells/ml after storage for 14 days.

Also, the two Bifidobacterium longum samples failed to sustain viablecount.

The Bifidobacterium breve YIT 4065 (FERM BP-6223) of the presentinvention sustained a viable count of 1×10⁸ cells/ml or more afterstorage, and provided fermented soymilk of excellent viable count andexcellent survival rate.

Example 2

Soymilk (product of Shikoku Kakoki, solid content 12.0%, crude fat2.78%, crude protein 5.03%) (7 L) was placed in a 10-L jar fermenter andsterilized with steam at 100° C. for 90 minutes.

Subculture was carried out several times by use of soymilk producedunder anaerobic conditions, to thereby produce Bifidobacterium breveinocula. Each inoculum was inoculated in an amount of 0.5%, and cultureduntil the pH reached 5.3.

After addition of a syrup solution (1.5 L) containing aspartame (0.13%)and citric acid (1.1%) to the fermented product in an amount of 5%, themixture was stored at 10° C. under anaerobic conditions. Viable cellswere counted and flavor was investigated, and the results are shown inFIG. 2.

The Bifidobacterium breve YIT 4065of the present invention sustained aviable count of 1×10⁸ cells/ml or more after storage at 10° C. for 14days, and the fermented soymilk produced by use of the same hadexcellent flavor.

INDUSTRIAL APPLICABILITY

The Bifidobacterium breve of the present invention exhibits an initialviable count of 1×10⁹ cells/ml or more and sustainment of a viable countof 1×10⁸ cells/ml or more after storage at 10° C. for 14 days, and thusis a remarkably excellent bifidobacterium which contributes to humanhealth through improvement of the intestinal environment.

The fermented soymilk employing the Bifidobacterium breve of the presentinvention contains at high concentration a bifidobacterium which mayhave a variety of possible uses, and advantageously sustains viablecount during storage without drastic reduction in the viable count.

What is claimed is:
 1. A biologically pure culture of a Bifidobacteriumbreve strain FERM BP-6223.
 2. A fermented soymilk comprising theBifidobacterium breve as recited in claim
 1. 3. The fermented soymilk ofclaim 2 further comprising one or more additives selected from the groupconsisting of sucrose, fructose, glucose, invertose, meat extract,peptone, and yeast extract.
 4. The fermented soymilk of claim 2 furthercomprising one or more acids selected from the group consisting ofcitric acid, malic acid, ascorbic acid, lactic acid and acetic acid. 5.The fermented soymilk of claim 2 further comprising one or moreadditives selected from the group consisting of saccharides, proteins,fats, oils, vitamins, plant extract, animal extract, bacterium extract,perfume and colorant.
 6. The fermented soymilk of claim 2 furthercomprising a microorganism selected from the group consisting ofLactobacillus, Streptococcus, Leuconostoc, Bacillus, Acetobacter,Gluconobacter, Saccharomyces, Candida, Rhodotorula, Pichia,Schizosaccharomyces, Torula, Zygosaccharomyces, Aspergillus, Eurotium,Monascus, Mycol, Neurospora, Penicillium, and Rhizopus.
 7. A method ofproducing a fermented soymilk comprising fermenting soymilk with theBifidobacterium breve strain of claim
 1. 8. The method of claim 7, whichfurther comprises adding one or more additives selected from the groupconsisting of sucrose, fructose, glucose, invertose, meat extract,peptone, and yeast extract.
 9. The method of claim 7, which furthercomprises adding one or more acids selected from the group consisting ofcitric acid, malic acid, ascorbic acid, lactic acid and acetic acid. 10.The method of claim 7, which further comprises adding one or moreadditives selected from the group consisting of saccharides, proteins,fats, oils, vitamins, plant extract, animal extract, bacterium extract,perfume and colorant.
 11. The method of claim 7, which further comprisesadding a microorganism selected from the group consisting ofLactobacillus, Streptococcus, Leuconostoc, Bacillus, Acetobacter,Gluconobacter, Saccharomyces, Candida, Rhodotorula, Pichia,Schizosaccharomyces, Torula, Zygosaccharomyces, Aspergillus, Eurotium,Monascus, Mycol, Neurospora, Penicillium, and Rhizopus.